Antral follicugenisis Flashcards
What is the theca and what are its functions?
- An envelope of connective tissue
- differentiates into theca interna & externa containing vascular tissue, immune cells and matrix factors
- Theca is critical for maintaining structural integrity of follicle and delivering nutrient to avascular GC layer
- Has LH receptors
What is important for preantral to antral progression?
Formation and differentiation of theca
Why is the theca important for preantral to antral progression?
- Neo-angiogenesis from theca, hence follicle interaction with systemic endocrine factors
- Acquisition of steroidogenic function as theca is steroidogenic – theca cells make androgens
How many sources do the theca cells derive from and what are they?
from 2 different sources in the embryonic gonad
- Mesenchymal (from mesonephros) cells become steroidogenic cell
- Stromal cells (indigenous to medullary region) become fibroblasts, perivascular smooth muscle cells and interstitial ovarian tissue
What is the antrum?
a single, large, fluid-filled cavity:
o Contains follicular fluid formed as exudate of plasma containing secretory products of oocyte & GC
How does antrum formation happen?
- When the follicle reaches a diameter of 200-400µm, surrounded by a vascularized theca, it becomes subjected to circulating influences.
- Fluid-filled spaces appear between the granulosa cells which soon coalesce together to form a single, large, fluid-filled cavity.
- As the fluid volume increases the follicle continues to expand greatly in size.
What is important for antrum formation?
o KL and Cx37 essential for antrum formation in lab animals – as k/o of these genes result in no antral follicles at all
What is the follicular fluid formed of?
- formed by filtration of thecal blood, its composition is different from plasma as contains secretory products of oocyte and granulosa cells
- As the fluid volume increases the follicle continues to expand greatly in size.
- Granulosa cells and Follicular fluids both increase as the follicle grows and develops
What is the theca externa?
- Concentrically arranged smooth muscle cells
- innervated by autonomic nerves
- have lymphatic vessels
- important during ovulation
What is the theca interna?
- Steroid producing cells
- Contain LH-R and insulin-R
- is richly vascularised
What is COC (cumulus-oocyte complex)?
an oocyte surrounded by specialized granulosa cells, called cumulus cells
- they interact with oocytes via gap junctions
- Have no LH-R
What happens to COC during the LH surge?
COC become expanded and have a sticky mucified nature as a response to the LH surge
How is COC expanded?
GC produce EGF-like ligands that bind LH and allow for secretion of hyaluronan and a complex of hyaluronan cross-linking proteins that cause expansion of COC
Why is the intercycle rise of FSH important?
- crucial for the recruitment of antral follicles into the menstrual cycle’
- needed for the selection of antral follicles
- needed for the selection of the dominant follicle
What is the two-cell, Two-Gonadotrophin Concept?
- The HPG axis acts to control antral follicle growth at this stage
- In response to LH, theca expresses key steroidogenic enzymes to make androgens from cholesterol – those enzymes are crucial
- Likewise granulosa cells respond to FSH by up-regulating aromatase and 17β-HSD to make oestrogens
What stimulates and maintains the expression of FSH-r?
Activin
Effects of FSH in the dominant follicles
- increase in granulosa cell proliferation
- increase in aromatase activity
- induce and maintain FSHr
- induce and maintain LHr
- interact with paracrine factors
What else interacts with FSH?
AMH and inhibin B
What kind of receptor is FSH-r?
G-protein coupled receptor
What happens when FSH binds to FSH-r?
- It causes a downstream cascade signalling pathway
- produces cAMP to activate PKA
- leads to differentiation of granulosa cells and increase follicular fluid, gap junctions, LHr, FSHr and P450 aromatase
- FSH gives positive feedback – stimulates FSHr genes, its aided by activin
What is the effect of androgens acting on granulosa cells?
Androgens act on granulosa cells (paracrine interaction) to upregulate androgen receptor & FSHR
What does AMH do in the dominant follicle?
• AMH (produced by GC of small antral follicles) acts as a brake on FSH recruitment of antral follicles by:
o decreasing FSH sensitivity
o decreasing FSH-stimulated aromatase expression
Why is counterbalancing the effects of AMH, FSH and androgens important?
to ensure against
o premature depletion of PF pool and/or
o premature selection of follicles by FSH
How is the dominant follicle recruited?
Each follicle has a different amount of FSH required to recruit one follicle and the follicle with the lowest threshold will be recruited within that environment
What will a dominant follicle have which helps it get recruited?
o lower threshold of FSH required
o Increased numbers of FSH-R
o FSH-r coupled more effectively to down-stream signalling – exert FSH actions more effectively
What are the actions of FSH?
• Growth and Oestradiol production
o increases cell division
o Increases in size
o increased aromatase
- Dominant follicle selection
- Increases area of theca vasculature – more open to circulating influences like insulin, growth factors and gonadotrophins
- FSH acts on the selected follicle to switch on the LHr gene
How does the dominant follicle survive the fall in FSH
- has increased sensitivity to FSH»_space;> increased FSH receptors
- has increased numbers of granulosa cells so more oestradiol
- acquisition of LH receptors - the LHR gene is switched on by FSH
- possible involvement of insulin-like growth factors 1&2 (IGF-2 particularly important in humans)
What does IGF-2 do in animals?
• FSH effects; stimulates androgen output and hence oestrogen
What suppresses IGF activity?
IGFBP (IGF-binding protein)
How is IGF made?
- IGF cleaved from IGFBP by PAPP-A (pregnancy-associated plasma protein A)
- PAPP-A expression high in DF
- Thought that other AF in cohort may have higher levels of IGFBP hence preventing co-stimulatory effect of IGF & FSH
What happens to the dominant follicle in the pre-ovulatory phase?
- Grows a lot
- Increase in follicular fluid and granulosa cells
What happens to the rest of the follicles that were not selected?
They die off
What is the role of LH in Antral Follicles?
- LH acts on theca and on the granulosa only after DF has been selected
- Induces progesterone receptors in the granulosa of the DF
- Increases the functions of the theca enzymes
- increases growth and steroidogenesis in dominant follicles
- Causes expansion of COC
- causes withdrawal of gap junctions between gc and oocyte leading to the resumption of meiosis
What does LH binding to LH-r in the theca interna do?
- LHr is a GPCR, it makes cAMP
- leads to the formation of steroids from cholesterol through the activation of enzymes that catalyse the steroid formation
What increases the actions of LH?
Insulin
What do LH and FSH have in common?
FSH have same 2nd messenger - cAMP
How does the cell distinguish between cAMP produced by LH and FSH?
- FSH produces low cAMP levels
- LH produces high cAMP levels
- linked to the difference in density of FSHr & LHr (LHr is more thought to be more prevalent than FSHr or LHr more effectively coupled to cAMP generation)
What else will be needed to support follicle growth?
- Angiogenic factors stimulated by primarily by androgens but also oestrogens – theca, gc, stroma all involved
- Basic fibroblast growth factor (bFGF) - it’s a Mitogen for endothelial cells which form the blood vessels, most potent angiogenic factor
- Vascular endothelial growth factor (VEGF) - it’s a mitogen for endothelial cells which form the blood vessels, enhances vascular permeability
- Ovarian lymphatic vessels recruited to theca and stroma layers around growing follicle, under control of VEGF-R3
Why do we need angiogenesis?
Constant re-modelling to allow for growth of follicle (2-20mm) through the ovarian tissue, angiogenesis of CL, tissue repair etc.
How do androgens act on the DF?
- Androgen binds to its (nuclear) receptors
- they translocate to the nucleus and bind to ARE (androgen response elements) on the HIF-1 gene
- this indues HIF-1 expression and that binds to HIF response elements (HRE) on the VEGF gene and that induces production of VEGF
- VEGF binds to receptors on the endothelial cells and induces proliferation
When is AMH secretion maximal?
- during small antral follicle stages and decreases to undetectable levels later
- Hence serum AMH reflect small AFs
What do low numbers of antral follicles signify?
• Low numbers of antral follicles are a sign of ovarian ageing – antral follicle count and AMH serum levels checked for patients that can’t conceive
How can AMH be used to predict fertility?
• Hence serum AMH reflect small AFs
• The number of antral follicles (AFC) in the early follicular phase correlates with numbers of growing follicles only
• This change in follicles and AMH is observable earlier than a rise in FSH serum level
•
However in women with PCOS, AMH levels are high but they’re still less fertile
What is used to determine the functional ovarian reserve?
AFC & serum markers FSH, AMH, E2 and Inhibin B used to determine “functional ovarian reserve”, but does not indicate true PF reserve ie what you are born with
What is Premature Ovarian Failure/Primary Ovarian Insufficiency?
- POI is clinical term for premature ovarian failure
- Defined as ovarian dysfunction <40yrs → oligomenorrhoea or amenorrhoea
- Overarching feature is infertility resulting from accelerated depletion or reduced follicle reserve.
What can cause POI?
o Environmental genotoxins induce DNA damage
o Mutations in genes like BRCA1/2
o Altered hormonal signalling
o Chromosomal defects e.g. Turner’s syndrome (XO) → have streak ovaries
o Autoimmune diseases including thyroiditis & Addison disease
treatments for POI
cryopreserving ovaries
biopsies
egg donations
How many types of 3D culture systems exist for follicles?
2 types: o Collagen o Alginate (product of seaweed) – able to produce antral follicles from small secondary preantral follicles in human and mokeys
requirement of a 3D culture systems for follicles?
- Hydrogel matrix to support follicle so that contact maintained between oocyte and gc
- Also to be permeable to media but also rigid to support the follicle
- Modifiable in terms of rigidity as follicle needs space to grow
